Deep Learning-Assisted Repurposing of Plant Compounds for Treating Vascular Calcification: An In Silico Study with Experimental Validation

Background. Vascular calcification (VC) constitutes subclinical vascular burden and increases cardiovascular mortality. Effective therapeutics for VC remains to be procured. We aimed to use a deep learning-based strategy to screen and uncover plant compounds that potentially can be repurposed for managing VC. Methods. We integrated drugome, interactome, and diseasome information from Comparative Toxicogenomic Database (CTD), DrugBank, PubChem, Gene Ontology (GO), and BioGrid to analyze drug-disease associations. A deep representation learning was done using a high-level description of the local network architecture and features of the entities, followed by learning the global embeddings of nodes derived from a heterogeneous network using the graph neural network architecture and a random forest classifier established for prediction. Predicted results were tested in an in vitro VC model for validity based on the probability scores. Results. We collected 6,790 compounds with available Simplified Molecular-Input Line-Entry System (SMILES) data, 11,958 GO terms, 7,238 diseases, and 25,482 proteins, followed by local embedding vectors using an end-to-end transformer network and a node2vec algorithm and global embedding vectors learned from heterogeneous network via the graph neural network. Our algorithm conferred a good distinction between potential compounds, presenting as higher prediction scores for the compound categories with a higher potential but lower scores for other categories. Probability score-dependent selection revealed that antioxidants such as sulforaphane and daidzein were potentially effective compounds against VC, while catechin had low probability. All three compounds were validated in vitro. Conclusions. Our findings exemplify the utility of deep learning in identifying promising VC-treating plant compounds. Our model can be a quick and comprehensive computational screening tool to assist in the early drug discovery process.

Association between bone mineral metabolism and vascular calcification in end-stage renal disease

Background Development of vascular calcification is accelerated in patients with end-stage renal disease. In addition to traditional risk factors of cardiovascular disease (CVD) abnormal bone and mineral metabolism together with many other factors contribute to the excess cardiovascular burden in patients on dialysis. Aortic calcification score and coronary calcification score are predictive of CVD and mortality. The aim of this study was to evaluate the possible relationship between arterial calcification and bone metabolism. Methods Thirty two patients on dialysis were included. All patients underwent a bone biopsy to assess bone histomorphometry and a 18F-NaF PET scan. Fluoride activity was measured in the lumbar spine (L1 – L4) and at the anterior iliac crest. Arterial calcification scores were assessed by computerized tomography for quantification of coronary artery calcification score and lateral lumbar radiography for aortic calcification score. Results This study group showed high prevalence of arterial calcification and 59% had verified CVD. Both CAC and AAC were significantly higher in patients with verified CVD. Only 22% had low turnover bone disease. There was a weak association between fluoride activity, which reflects bone turnover, measured in the lumbar spine, and CAC and between PTH and CAC. There was also a weak association between erosion surfaces and AAC. No significant association was found between calcification score and any other parameter measured. Conclusions The results in this study highlight the complexity, when evaluating the link between bone remodeling and vascular calcification in patients with multiple comorbidities and extensive atherosclerosis. Several studies suggest an impact of bone turnover on development of arterial calcification and there is some evidence of reduced progression of vascular calcification with improvement in bone status. The present study indicates an association between vascular calcification and bone turnover, even though many parameters of bone turnover failed to show significance. In the presence of multiple other factors contributing to the development of calcification, the impact of bone remodeling might be diminished. Trial registration The study is registered in ClinicalTrials.gov protocol registration and result system, ID is NCT02967042. Date of registration is 17/11/2016.

Lipoxin Alleviates Diabetic Vascular Calcification via the YAP Pathway

Background: Vascular calcification is highly prevalent in patients with diabetes and has detrimental consequences. However, no effective prevention and treatment methods are currently available. Extensive evidence has demonstrated the protective effect of lipoxin (LX) against vascular diseases. However, whether LX prevents diabetic vascular calcification remains unknown. Here, we tested the hypothesis that LX alleviated osteogenic differentiation and subsequent calcification of vascular smooth muscle cells (VSMCs).Methods: In vitro, human aortic smooth muscle cells (HASMCs) were incubated in osteogenic medium (OM) with advanced glycation end products (AGEs) and LX to further determine the underlying mechanisms. An in vivo diabetic mouse model was established using a combination of a high-fat diet and multiple formulations of low-dose streptozotocin (STZ). Cell culture, alkaline phosphatase (ALP) staining, ALP activity, Alizarin red staining, von kossa staining, determination of calcium content, western blot analysis, immunohistochemistry, and immunofluorescence staining and statistical analysis were used in our study. Results: AGEs dose-dependently induced calcification and expression of osteogenesis-related markers, including Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and type I collagen (COL1), coupled with the activation of yes-associated protein (YAP). Mechanistically, YAP activation enhanced the AGE-induced osteogenic phenotype and calcification, but inhibition of YAP signalling alleviated this trend. Consistent with the in vitro results, diabetes promoted YAP expression as well as the subcellular localisation of the protein in the nucleus in the arterial tunica media. Interestingly, treatment with LX reduced vascular osteogenesis and calcification in diabetic mice, which was correlated with the reduced YAP levels. In addition, LX significantly inhibited COL1 accumulation and modulated the extracellular matrix. Our results further demonstrated that a pharmacological agonist of YAP reversed LX-mediated protection against osteogenic phenotypic conversion and calcification in VSMCs.Conclusions: These results demonstrate that LX attenuates transdifferentiation and calcification of VSMCs in diabetes mellitus via the YAP signalling axis, suggesting that LX is a potent therapeutic strategy to prevent diabetic vascular calcification.

Vascular Calcification: Key Roles of Phosphate and Pyrophosphate

Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.

The Relationship between Serum Sclerostin Levels and Bone Mineral Disorders and Vascular Calcification in Hemodialysis Patients

Background and aim of the study: Sclerostin is produced by osteocytes and has been shown to down-regulate the synthesis of many markers of bone formation by osteogenic cells. The aim of this study to investigate the relationship between serum sclerostin levels and bone mineral disorders and vascular calcification in hemodialysis patients (HD). Methods:This is a cross-sectional study of 70 patients with ESRD on regular HD for at least six months, Theodor Bilharz Research Institute, Giza, Egypt.Twenty-five subjects who matched the ages, genders, and demographics of the study patients were included as a control group.All patients and control groups included in the study underwent a full through history and clinical examination. Serum calcium, phosphorus, alkaline phosphatase and intact PTH (iPTH) levels were measured. Serum sclerostin was measured by an ELISA. Bone Mineral Densitometry Measurements BMD (g/cm2) was determined by dual-energy X-ray absorptiometry (DXA). CT scan was done to detect the presence or absence of vascular calcification and transthoracic echocardiogram to detect the presence or absence of valvular calcification. Results:The mean seumscleostin levels was a statistically significant high in the HD patients when compared with the control group (156.8 ±121.4 Vs.29.38±0.84, p =0.0001 ) and statistically significant high mean ALP in the HD patients when compared with the control group (147.2 ± 94.3 Vs. 38.8 ±23.4, p = 0.0001). The mean BMD was statistically significant low in the HD patients when compared with the controls (0.839±0.086 g/ m2 Vs.1.306 ±0.153 g/ m2, p = 0.0001).The mean seumscleostin levels was statistically significant high in the HD patients with vascular and valvular calcification when compared with HD patients without calcification.Using spearman correlation coefficient analysis, there was statistically significant negative correlations between serum sclerostin levels and iPTH(r=-0.362, p =0.0021), ALP (r=-0.301, p =0.0114), and BMD (r=-0.469, p =0.0278 ), and there was a statistically significant positive correlation between serum sclerostin levels and phosphate(r=0.5829, p =0.0001 ).Independent predictors of BMD in HD patients were determined using multi-variate regression analysis. Sclerostin levels, iPTH, ALP, and age were found to be independent predictors of BMD. Conclusion: High sclerostin levels in patients with ESRD on HD were associated with high risk of vascular and valvularcalcification and were independent predictors of low BMD in such population.

Unspliced XBP1 Counteracts β-catenin to Inhibit Vascular Calcification

Background: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the unspliced (XBP1u) and spliced (XBP1s) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. Methods: We aim to investigate the role of XBP1u in vascular calcification.XBP1u protein levels were reduced in high phosphate (Pi)-induced calcified vascular smooth muscle cells (VSMCs), calcified aortas from mice with adenine diet-induced chronic renal failure (CRF) and calcified radial arteries from CRF patients. Results: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers runt-related transcription factor 2 (Runx2) and msh homeobox2 (Msx2), and exacerbated high Pi-induced VSMC calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high Pi-induced VSMCs significantly inhibited osteogenic differentiation and calcification. Consistently, SMC-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u bound directly to β-catenin, a key regulator of vascular calcification, via aa 205-230 in its C-terminal degradation domain. XBP1u interacted with β-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited β-catenin/T-cell factor (TCF)-mediated Runx2 and Msx2 transcription. Knockdown of β-catenin abolished the effect of XBP1u deficiency on VSMC calcification, suggesting a β-catenin-mediated mechanism. Moreover, the degradation of β-catenin promoted by XBP1u was independent of glycogen synthase kinase 3β (GSK-3β)-involved destruction complex. Conclusions: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting β-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of β-catenin and a promising target for vascular calcification treatment.